Insulin, a peptide secreted by the pancreatic beta cells, plays a central role in the control of blood glucose levels in the body. When insulin is not properly secreted or the secreted insulin does not work in the body, the blood glucose level is not regulated, and thus diabetes occurs. This diabetes is called type II diabetes. Type I diabetes is caused when the pancreas does not make enough insulin to increase the blood glucose level.
Type II diabetes is usually treated with oral hypoglycemic agents chemically synthesized, and in some cases, patients are treated with insulin. Meanwhile, type I diabetes requires insulin treatment.
The insulin treatment method currently used is injection of insulin before/after meals. However, such insulin injection should be continuously administered three times per day, which causes pain or discomfort to the patients. There have been various attempts to overcome the problem. One of them is a method of delivering a peptide drug via oral or nasal inhalation by improving its membrane permeability. Undesirably, the method showed very low delivery efficiency, compared to the injectable formulations, and thus there are still many difficulties in maintaining in vivo activity of the peptide drug at the required level.
Meanwhile, there was a method of delaying drug absorption after a subcutaneous injection of a large amount of the drug, so as to maintain the blood level by only one daily injection. Some of the developed drugs (Lantus, Sanofi-aventis) were approved, and are now used for the patients. In addition, studies have been conducted to prolong the action, leading to development of Levemir (Novo Nordisk) prepared by modification of insulin with fatty acid, in which the protracted action occurs through self-association of insulin molecules at the site of injection and through reversible binding to albumin in the blood. However, these methods generate pains at the site of injection, and the daily injections also cause considerable discomfort to the patient.
Many efforts have been made to improve the serum stability of peptide drugs and maintain the drugs in the blood at high levels for a prolonged period of time, thereby maximizing the pharmaceutical efficacy of the drugs. These long-acting formulations of peptide drugs need to increase the stability of the peptide drugs and maintain the titers at sufficiently high levels without causing immune responses in patients. For the preparation of the long-acting formulations of peptide drugs, a polymer having high solubility, such as polyethylene glycol (PEG), was conventionally used to chemically modify the surface of a peptide drug.
PEG non-specifically binds to a specific site or various sites of a target peptide to give an effect of increasing the molecular weight of a peptide, and thus inhibiting the loss by the kidney, and preventing hydrolysis, without causing any side-effects. For example, WO 2006/076471 describes that a B-type natriuretic peptide (BNP), which binds to NPR-A to activate the production of cGMP and leads to reduction in the arterial blood pressure, and as a result, is used as a congestive heart failure therapeutic agent, is linked to PEG, thereby sustaining its physiological activity. U.S. Pat. No. 6,924,264 describes that PEG binds to the lysine residue of an exendin-4 to increase its in-vivo residence time. This method increases the molecular weight of PEG, and thus increases the in-vivo residence time of the peptide drug. However, as the molecular weight is increased, the titer of the peptide drug is remarkably reduced, and the reactivity with the peptide is also reduced. Accordingly, it undesirably lowers the yield.
WO 02/46227 describes a fusion protein prepared by coupling GLP-1, an exendin-4, or an analog thereof with human serum albumin or an immunoglobulin fragment (Fc) using a genetic recombination technology. U.S. Pat. No. 6,756,480 describes an Fc fusion protein prepared by coupling a parathyroid hormone (PTH) and an analog thereof with Fc region. These methods can address the problems such as low pegylation yield and non-specificity, but they still have a problem in that the effect of increasing the blood half-life is not as noticeable as expected, and in some cases, the titers are also low. In order to maximize the effect of increasing the blood half-life, various kinds of peptide linkers have been used, but there is a possibility of causing an immune response. Further, if a peptide having disulfide bonds, such as BNP, is used, there is a high probability of misfolding, and if a peptide having non-naturally occurring amino acid residues is used, it can be produced by genetic recombination only with great difficulty.